Biochemical studies of mammalian skeletal muscle have found that the major contractile protein myosin undergoes transitions in its subunit structure during development from its embryonic to its adult forms. We will investigate the physiological function(s) of these various forms, or isoenzymes, of myosin in rabbit and avian skeletal muscles. This will be done through the use of single muscle fiber preparations from which the surface membranes have been removed, thereby allowing direct control of muscle activation with Ca2+. Measurements of tension, stiffness and shortening velocity, as well as the tension transients in response to rapid length changes, will be made in order to determine the mechanical properties of the myosin cross-bridges and the kinetics of interaction of myosin with actin. These same measurements will also be made in living single fibers obtained from muscles at different stages of development in order to determine the extent to which developmental changes in mechanical properties are due to changes in the contractile proteins per se. The myosin isoenzymes composition of these same skinned and living fiber preparations will then be determined using SDS polyacrylamide gel electrophoresis and high voltage iso-electric focusing procedures that have been developed in our laboratory for use on a micro-scale. These mechanical and biochemical measurements will be done on muscles that are destined to be fast and those that are destined to be slow in the adult and will encompass the fetal, neonatal and adult stages of development. Such measurements made on the same skinned single fiber preparations will allow straightforward conclusions regarding the possible relationships between the myosin isoenzymes that are present and physiological function. This experimental approach will provide new and valuable information about the molecular basis of the regulation of the interaction of myosin with actin in preparations which retain the structural filamentary organization of living muscle and should therefore further our understanding of muscle contraction.